Interferons (IFNs) belong to the class of cytokines which allow communication between cells to trigger the protective defenses of the immune system. IFNs are made and released by lymphocytes in response to the presence of pathogens like bacteria, viruses, and even tumor cells. IFN is produced and secreted by various mammalian cell lines when infected by pathogens and constitutes an important player in innate immunity against these pathogens. It also constitutes a significant therapeutic molecule in a number of viral diseases and cancers. There are hundreds of virus and IFN-stimulated genes and although their promoters harbor specific core sequences elements, they have context heterogeneity, reiterations, and different transactivation potential. These differences may account for responses to different types of IFN and viruses.
The transcription of IFN genes themselves is mediated via specific virus response elements (VRE) that bind different IFN response factors (IRF) such IRF-3 and IRF-7 in the promoters of IFN genes (Paun et al, 2007).
IFN induces the STAT/JACK pathway leading to activation and binding of transcriptional activators to the interferon-stimulated response element (ISRE) in the promoters of the IFN-stimulated genes (Sato et al., 2001; Borden et al., 2007).
There are hundreds of virus- and IFN-stimulated genes that exist in the human genome (Khabar et al., 2004) and although their promoters harbor specific core sequences elements, they have context heterogeneity, variable reiterations, and different transactivation potential. These differences may account for responses to different types of viruses, IFNs, and IRFs.
Yet, it is not possible to detect differential expression of IFN genes and IFN-stimulated genes with a versatile, simple, and sensitive method and assaying IFN bioactivity and potency is largely made with antiviral assay that requires virus propagation, virus stock maintenance, and cumbersome steps. Additionally, current gene reporter assays lack sensitivity and specificity. So, in general, the bioactivity of interferon, necessary for evaluation of therapeutic IFNs and for diagnostic purposes, is assayed customarily by a viral cytopathic effect assay or by other assays that require multiple steps such as cell destruction or dye incorporation. Certain assays utilize mRNA expression levels. The amount of transcripts of these genes can be assessed by quantitative real-time PCR of extracted mRNA. However, this method requires a large number of experimental steps, including cell lysis, RNA extraction, amplification steps which may lead to inaccurate quantification of mRNA levels. As a consequence, it may sometimes not be possible to detect differences of the expression pattern or to distinguish the trigger of the stimulation of IFN-stimulated genes, i.e. to distinguish the type of IFN or type of pathogen.
Although, there are existing reporter assays utilizing IFN-inducible promoters or standard IFN stimulated response elements, but they suffer from a lack sensitivity and selectivity.
Though the antiviral bioassay is a method of choice for titrating IFN in biological samples, this approach has been challenged with several alternatives in order to over comes these limitations. As an example, Lleonart et al (1990) developed MxA/hGH reporter assay to quantify type I IFN on Vero cells. The construct of the human growth hormone (hGH) placed under control of human IFN inducible MxA promoter which transfected into African Green monkey kidney cells (Vero cells). The production of hGH is measured by a hGH-specific radio-immunoassay (Canosi et al., 1996). However, substituted hGH gene with Luciferase gene transfected in Vero cells and the activity of Luciferase accumulated in Vero cells can be read directly after cell lysis. In recently described modified example of a reporter gene assay (Fray, Mann, and Charleston, 2001), the human Mx promoter is linked to a chloramphenicol acetyltransferase (CAT) reporter. Mx/CAT reporter was transfected into Madin-Darby Bovine Kidney (MDBK) cells and CAT expression was quantitated by commercially available ELISA. Furthermore, it was assumed that CAT reporter assay is accurate since its CAT gene is not present in eukaryotic system. This should eliminate possibility of interference to the system by indigenous proteins (Fray, Mann, and Charleston, 2001). Certain commercial reporter constructs (Stratagene, SA biosciences) are available in which tandem repeats of classical ISRE sequences (AGTTTCACTTTCCC (nucleotides 32-45 of SEQ ID NO:61)) exist of known IFN-stimulated genes, but, they lack desired sensitivity and selectivity. For example, ED50 of those constructs only ranged from 250-300 IU/m.
Thus, the object of the present disclosure is to provide a simplified and more differential approach to different types of IFN and viruses.